Binding Kinetics between Membrane-Bound Kinesin Motors and Microtubules

Abstract

Despite the vital role that kinesin plays in transporting membraneous organelles in eukaryotic cells, there is a knowledge gap between our understanding of the chemomechanical cycle of single motors and the dynamics of multi-motor-driven vesicle transport in vivo. One of the fundamental mechanisms that remain obscure is how a lipid bilayer affects the binding kinetics between kinesin and microtubules. We set up a 2D system in which kinesin-1 motors were attached to a supported lipid bilayer to monitor the ensemble kinetics of motor binding to microtubules under total internal reflection fluorescence (TIRF) microscope. Freely diffusing kinesin-1 motors rapidly accumulated onto the microtubule upon its landing and the accumulation process reached steady-state within seconds. The observed accumulation rate was found to scale linearly with motor density. Using a simple model of binding kinetics between diffusing kinesin motors and the bound microtubule, we calculated a bi-molecular on-rate constant of 1.2 x 10−4 (motors/μm2)−1s−1. When 30% cholesterol was incorporated into the lipid membrane, which lead to a 4-fold drop in diffusion coefficient, the on-rate constant of kinesin was not altered, meaning that the binding of motors to microtubules was not limited by membrane diffusion. We developed a stochastic computational model in which kinesin-1 motors freely diffused on a lipid bilayer and bound to the microtubule with a first-order on-rate constant when they were within the binding zone where microtubule was accessible. The computational simulation recapitulated the experimental data and the theoretical model well.